Taste buds are the transducing elements of gustatory sensation. As such, they represent the first step in the process by which nutritious substances, such as Na+ salts, sugars, and amino acids can be detected and distinguished from harmful substances, such as acids and toxic bitter compounds. Understanding the steps in this process may lead to therapeutic interventions that can be used to modulate food intake, a critical factor in controlling obesity. Each taste bud comprises three types of elongate taste cells and a population of basal cells. Understanding the functional role of each cell type is fundamental to understanding how each type of stimulus is detected and how this information is transmitted to the nervous system. Of the three types of fusiform cells, the Type II cells, which express the receptors and signaling effectors for bitter, sweet, and umami transduction, are the best understood. They release ATP to activate purinergic receptors on afferent nerve fibers. Type III, or presynaptic cells, detect sour stimuli and release serotonin and noradrenalin. Type I, or glial-like cells are the most abundant cells in the taste bud but also the least understood. Similar to glial cells in the nervous system, their membranes closely envelope other taste cells and they express NTPDase2, an ectoATPase that degrades ATP that is released from Type II cells. Recent data suggest that Type I cells may have additional functions, including transduction of Na+ salts and modulation of Type II cells. Unlike Type II and Type III cells, there are no fluorescent reporters for identification of Type I taste cells, making identification and functional characterization difficult. The proposed studies will utilize existing reagents and technology to develop a gene-targeted mouse that will express a nuclear-targeted fluorescent reporter (GFP) and Cre recombinase from the NTPDase2 promoter. Nuclear localization of GFP will insure that Type I cells can be distinguished from the other cell types, both within the bud and after isolation. Moreover, Cre recombinase will allow selective deletion of sequence from Type I taste cells, when these mice are crossed with mice carrying an appropriate floxed allele. In the first aim, we will validate the expression of the targeted allele, by crossing the mce to a commercially available Cre reporter line, Rosa26-tdTomato, which will express a red fluorescent reporter upon Cre-mediated excision of sequence flanked by loxp sites upstream of the reporter. In the second aim, we will utilize the mice to test the hypothesis that Type I cells are necessary for amiloride-sensitive salt taste. Whole cell recording will test whether Type I cells express functional amiloride- sensitive Na+ currents. Further, we will develop the methodology to specifically ablate type I taste cells, using the diphtheria toxin receptor targeted to Type I taste cells. In summary, these experiments will allow us define the Type I cell population and provide a new tool to dissect the functions of this most common taste cell type.

R21DC011890

2012-02-03

2015-12-31